CN103907596A

CN103907596A - Bdellovibrio particulate agent

Info

Publication number: CN103907596A
Application number: CN201210594026.4A
Authority: CN
Inventors: 黄艳
Original assignee: Qingdao Jinlianxin Trading Co Ltd
Current assignee: Qingdao Jinlianxin Trading Co Ltd
Priority date: 2012-12-30
Filing date: 2012-12-30
Publication date: 2014-07-09

Abstract

A bdellovibrio particulate agent is characterized by being prepared by: mixing plant oil, agar with a mass concentration of 5%-10%, and a bacteria liquid with a bdellovibrio content of 10<10>-10<11> CFU/mL according to a volume ratio of 3:50:100, then adding sodium carboxymethylcellulose with a mass concentration of 2% and mixing uniformly, dropwise adding the mixed solution into a CaCl2 solution with a mass percentage of 10%, solidifying for 12 h, and then performing freeze drying at -20 DEG C for 24 h on bdellovibrio solidified particles to obtain the particulate agent. The bdellovibrio particulate agent is high in mechanical strength, the bdellovibrio activity is well stored, and the bdellovibrio particulate agent is stable in chemical performances, low in cost and long in storage time, and is applicable to multiple industries such as aquatic product culture, livestock and poultry culture and the like.

Description

A kind of Bdellovibrio granule

Technical field

The present invention relates to a kind of microorganism formulation, specifically a kind of bacteriophagic Bdellovibrio granule.

Background technology

Bacteriophagic Bdellovibrio (Bdellovibrio bacteriovorus) (hereinafter to be referred as Bdellovibrio) is a kind of parasitism bacterium of making a living with predator bacteria specially, there is research to point out, Bdellovibrio purifying aquatic water water quality, control or the generation that reduces aquatic animal disease with popular on effect remarkable, in breeding process, add and introduce Bdellovibrio and can play disease prevention and cure, improve survival rate, increase the effect of weight and body length and purifying aquaculture ecotope.Chen Liyun etc. study discovery, after interpolation Bdellovibrio, can make the incidence of the red mouth disease of turbot drop to 0.93% by 10.17%, illustrate that Bdellovibrio can effectively prevent the generation of red mouth disease, in addition, aspect the red mouth disease for the treatment of, can the red mouth disease of 100% healing after interpolation Bdellovibrio.Application Bdellovibrio control aquatic livestock bacterial disease and sterilization have good development prospect, the bdellovibrio bacteriovorus preparation of Chinese commodity is mainly aqua at present, also has a small amount of freeze dried powder, Bdellovibrio aqua because of the holding time short, factors such as the unstable and transportation inconvenience of quality and affected its result of use, freeze dried powder has also limited its application to a great extent because production cost is high, and microbial immobilized method can be positioned free microorganism the area of space limiting, and keep active, and can recycle, especially embedded immobilization method, because of simple to operate, little on microbial activity impact in preparation process, the granule strength of preparation is higher, deeply be subject to especially probiotics research staff's favor.

Immobilized microorganism technique is that elite microorganism is fixed on the carrier of choosing, makes its highly dense and keeps biologically active, and the biotechnology that can breed fast, in a large number under optimum conditions, the most conventional with investment.Its principle is that biological cell is trapped in water-insoluble embedded material network hole, and its network can stop the leakage of cell, can allow matrix infiltration and product diffuse out simultaneously.Embedded material can be divided into two large classes: (l) natural macromolecule amylose class, as alginate, agar, gelatin etc., wherein maximum with sodium alginate and carragheen application, they have solidifies conveniently, little and the immobilization density advantages of higher to Ecotoxicology, but their Resistance to microbes poor-performings, mechanical strength is low, but can use crosslinking agent to carry out stabilization processes, but vigor and mass-transfer performance can decline again.(2) synthetic macromolecular compound, as polypropylene phthalein amine, polyvinyl alcohol (PvA) etc.The outstanding advantages of this class crosslinking agent is that Resistance to microbes performance is good, and mechanical strength is high, stable chemical performance.But the formation condition of polymer network is more violent, larger to the infringement of microbial cell, and also the diversity and the controllability that are shaped are bad.Zhang little Neng etc. have studied the optimization of bacteriophagic Bdellovibrio granule preparation condition in " optimization of bacteriophagic Bdellovibrio granule preparation condition " by sodium alginate to embed method, because sodium alginate price is higher, the Bdellovibrio granule cost making is higher, is not suitable for large-scale industrial production.

Summary of the invention

It is lower that technical problem to be solved by this invention is to provide a kind of cost, the better bacteriophagic Bdellovibrio granules such as vigor and mechanical strength.

For solving the problems of the technologies described above, the technical solution used in the present invention is, a kind of Bdellovibrio granule, its for the agar and the Bdellovibrio content that are 5%-10% by vegetable oil, mass concentration be 10 ¹⁰-10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is mixed, then to add mass concentration be 2% sodium carboxymethylcellulose and mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, then by the Bdellovibrio cured granulate granule that freeze drying 24h makes under-20 ℃ of conditions.

Above-mentioned Bdellovibrio granule, agar and Bdellovibrio content that it is is 5% by peanut oil, mass concentration are 10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is mixed, then to add mass concentration be 2% sodium carboxymethylcellulose and mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, then by the Bdellovibrio cured granulate granule that freeze drying 24h makes under-20 ℃ of conditions.

Bacteriophagic Bdellovibrio granule mechanical strength of the present invention is high, and Bdellovibrio vigor is intact, stable chemical performance, and cost is low, and the holding time is long, can be for multiple industries such as aquaculture, livestock and poultry cultivation.

Embodiment

Embodiment 1

A kind of Bdellovibrio granule, agar and Bdellovibrio content that it is is 5% by peanut oil, mass concentration are 10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is mixed, then to add mass concentration be 2% sodium carboxymethylcellulose and mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, then by the Bdellovibrio cured granulate granule that freeze drying 24h makes under-20 ℃ of conditions.Its concrete preparation method is:

(1) preparation of Bdellovibrio bacterium liquid:

Bdellovibrio bacterial classification is inoculated in to 100 mL in 1/10 NB medium, adding 100 L concentration is 1.0 × 106CFU/mL Escherichia coli bacteria suspensions, shaking table shaken cultivation 48h under 100r/min at 35 ℃ of temperature, detects total plate count, and adjusting Bdellovibrio bacterial concentration is 1.0 × 10 ¹¹cFU/mL is for subsequent use.

(2) prepare Bdellovibrio embedded particles:

The agar that peanut oil, mass concentration are 5% and Bdellovibrio content are 10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is put into stirred vessel, then adding mass concentration is 2% sodium carboxymethylcellulose, 25 ℃ of constant temperature stir 30min, make it to mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, by Bdellovibrio cured granulate freeze drying 24h under-20 ℃ of conditions, make granule again.

Embodiment 2

A kind of Bdellovibrio granule, its concrete preparation method is:

(1) preparation of Bdellovibrio bacterium liquid:

Bdellovibrio bacterial classification is inoculated in to 100 mL in 1/10 NB medium, adding 100 L concentration is 1.0 × 106CFU/mL Escherichia coli bacteria suspensions, shaking table shaken cultivation 48h under 100r/min at 35 ℃ of temperature, detects total plate count, and adjusting Bdellovibrio bacterial concentration is 1.0 × 10 ¹⁰cFU/mL is for subsequent use.

(2) prepare Bdellovibrio embedded particles:

The agar that peanut oil, mass concentration are 10% and Bdellovibrio content are 10 ¹⁰the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is put into stirred vessel, then adding mass concentration is 2% sodium carboxymethylcellulose, 25 ℃ of constant temperature stir 30min, make it to mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, by Bdellovibrio cured granulate freeze drying 24h under-20 ℃ of conditions, make granule again.

Embodiment 3

Cured granulate viable count detects

Bdellovibrio granule of the present invention can be preserved by normal temperature, gets the normal temperature Bdellovibrio granule of different holding times, detects its number of viable, and the Bdellovibrio granule of preparing take embodiment 1 is experimental subjects.

Get Bdellovibrio granule 0.1g prepared by embodiment 1, add the sodium citrate solution of 10 mL 0.055 mol/L, after the complete disintegration of particle, mix, after gradient dilution, adopt the viable bacteria content of Bdellovibrio in the double-deck agar plate method detection of running water particle, finally calculate the number of viable of Bdellovibrio in every gram of particle.After testing, in the Bdellovibrio granule of preparation, viable bacteria concentration is from 1.0 × 10 in 6 months the holding time ¹¹cFU/mL is steadily down to 5 × 10 ¹⁰cFU/mL, reduction very steadily slowly, meet production requirement.

Claims

1. a Bdellovibrio granule, is characterized in that: agar and Bdellovibrio content that it is is 5%-10% by vegetable oil, mass concentration are 10 ¹⁰-10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is mixed, then to add mass concentration be 2% sodium carboxymethylcellulose and mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, then by the Bdellovibrio cured granulate granule that freeze drying 24h makes under-20 ℃ of conditions.

2. Bdellovibrio granule according to claim 1, is characterized in that: agar and Bdellovibrio content that it is is 5% by peanut oil, mass concentration are 10 ¹¹the ratio that the bacterium liquid of CFU/mL is 3:50:100 according to volume ratio is mixed, then to add mass concentration be 2% sodium carboxymethylcellulose and mix, it is in 10% CaCl2 solution, to solidify 12h that mixed liquor is splashed into mass fraction, then by the Bdellovibrio cured granulate granule that freeze drying 24h makes under-20 ℃ of conditions.